PROJECT SUMMARY The molecular mechanism of inhaled anesthetics in general anesthesia remains an important unanswered question in neuroscience and human health. Since the first demonstration of ether-induced anesthesia more than 160 years ago, theories of anesthesia have sought to understand the role of the membrane in anesthetic action. This application seeks to establishes the plasma membrane as a relevant target for inhaled anesthetics. Lipid membranes spontaneously partition into regions of ordered and discorded lipids known as lipid rafts. Palmitoylation of proteins drives the proteins into the ordered phase. We have shown that anesthetics disrupt these domains in live cell membranes, but disruption has not been linked to anesthetic ion channels. We will test the hypothesis that anesthetics disruption of lipid rafts releases lipid modifying enzymes to activate potassium channels through a chemical signal. Specifically, GM1 rafts sequester phospholipase D (PLD), disruption of the raft releases PLD allowing the enzyme to find its substrate and generate anionic lipid phosphatidic acid (PA). The PA then regulates the two-pore domain potassium channel (K2P) TREK-1. TREK- 1 is also an anesthetic sensitive channel. We aim to characterize the effects of anesthetics on lipid raft structure in the membrane. In a second aim, we will elucidate the mechanism of TREK-1 activation through anesthetic disruption of the membrane and in so doing establish the membrane as a bon a fide target of anesthetic action. In a third aim we will develop better imaging tools and a live cell fluorescent assay for monitoring raft disruption in cells.